Article
Canstatin as a potential therapeutic molecule for rheumatoid arthritis synovial fibroblast mediated altered angiogenesis
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Authors
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Corinna Heck
- Justus-Liebig-Universität Giessen, Campus Kerckhoff, Rheumatologie und Klinische Immunologie, Bad Nauheim
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Sophie Haun
- Justus-Liebig-Universität Giessen, Campus Kerckhoff, Rheumatologie und Klinische Immunologie, Bad Nauheim
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Daria Kürsammer
- Justus-Liebig-Universität Giessen, Campus Kerckhoff, Rheumatologie und Klinische Immunologie, Bad Nauheim
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Klaus Frommer
- Justus-Liebig-Universität Giessen, Campus Kerckhoff, Rheumatologie und Klinische Immunologie, Bad Nauheim
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Mona Arnold
- Justus-Liebig-Universität Giessen, Campus Kerckhoff, Rheumatologie und Klinische Immunologie, Bad Nauheim
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Markus Rickert
- University Hospital Giessen and Marburg, Dept. of Orthopaedics and Orthopaedic Surgery, Giessen
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Katrin Lips
- Justus-Liebig-Universität Giessen, Department of Experimental Trauma Surgery, Giessen
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Stefan Rehart
- Agaplesion Markus Hospital, Orthopaedics and Trauma Surgery, Frankfurt am Main
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Ulf Müller-Ladner
- Justus-Liebig-Universität Giessen, Campus Kerckhoff, Rheumatologie und Klinische Immunologie, Bad Nauheim
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Elena Neumann
- Justus-Liebig-Universität Giessen, Campus Kerckhoff, Rheumatologie und Klinische Immunologie, Bad Nauheim
| Published: | August 30, 2023 |
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Outline
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Introduction: Increased angiogenesis in the inflamed synovium of RA patients is a pathological feature in RA. Key players in RA progression are chronically activated RA synovial fibroblasts (RASF) promoting angiogenesis and degradation of matrix, cartilage and bone. Canstatin, a matrix-derived anti-angiogenic collagen IV fragment, blocks the angiopoietin (ANGPT)/Tie2 pathway in endothelial cells (EC).
Objective: Characterization of RASF-EC interactions with focus on vessel formation regarding the influence of canstatin and ANGPT2 expression.
Methods: RASF were repetitively stimulated thrice every 24h with 0.05 ng/µl IL-1β. 2D tube formation assay was performed using HUVEC (+/- prestimulated with 0.2 µg/ml canstatin for 20 h) and 15% RASF seeded on Matrigel®. RASF/HUVEC were treated with 0.5 µg/ml canstatin. Tube thickness and the area covered by the formed cellular network were measured. RNA was extracted from tubes and analysed by qPCR. Cartilage was subcutaneously co-implanted with RASF alone or with 0.5 µg/ml canstatin into SCID mice. Contralaterally, cartilage without RASF but with canstatin was implanted. Vessel formation was evaluated after 3–45 days.
Results: RASF stimulated once showed a significant IL-6, IL-11 and CXCL2 increase compared to unstimulated controls, while subsequent repetitive stimulation of RASF led to a significant decrease in IL-6, IL-11 and CXCL2 (p<0.004 each). RASF significantly reduced tube thickness (22.9 µm vs. 16.6 µm, p=0.014) and the network area (p<0.0001) and significantly increased ANGPT2 2-fold compared to HUVEC alone. Canstatin stimulation of HUVEC and HUVEC+RASF also led to disturbed tube formation with reduced tube thickness (e.g. 22.9 to 14.6 µm (SD=1.4, p<0.001), with a significant 1.6-fold decrease of ANGPT2. In SCID mice, RASF-mediated altered vessel formation leading to helix-like vessels was observed at early time points in implants with RASF (day 3–9). The addition of canstatin into implants resulted in a decrease of helix-like vessel formation.
Conclusion: Repetitive stimulation of RASF resulted in an inflammatory adjustment to IL-1β, leading to significant reduction of inflammatory and angiogenic parameters IL-6, IL-11 and CXCL2 suggesting an influence on vessel formation. RASF-mediated influence on altered angiogenesis was confirmed in the tube formation assay. ANGPT2 was demonstrated as a pathological vessel marker for RASF-mediated altered angiogenesis. Interestingly, the anti-angiogenic canstatin reduced the RASF-induced helix-like vessels in vivo.
